Hindawi_Gobby_et_al

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Hindawi_Gobby_et_al

Influence of Melatonin on the Proliferative and Apoptotic Responses of the Prostate
under Normal and Hyperglycemic Conditions.
Gobbo, Marina; Dizeyi, Nishtman; Abrahamsson, Per-Anders; Bertilsson, Per-Anders;
Masitéli, Viviane Sanches; Pytlowanciv, Eloisa Zanin; Taboga, Sebastião R; Góes, Rejane M
Published in:
Journal of Diabetes Research
DOI:
10.1155/2015/538529
Published: 01/01/2015
Link to publication
Citation for published version (APA):
Gobbo, M., Dizeyi, N., Abrahamsson, P-A., Bertilsson, P-A., Masitéli, V. S., Pytlowanciv, E. Z., ... Góes, R. M.
(2015). Influence of Melatonin on the Proliferative and Apoptotic Responses of the Prostate under Normal and
Hyperglycemic Conditions. Journal of Diabetes Research, 2015, [538529]. DOI: 10.1155/2015/538529
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Volume 2015, Article ID 538529, 18 pages
http://dx.doi.org/10.1155/2015/538529
Research Article
Influence of Melatonin on the Proliferative
and Apoptotic Responses of the Prostate under
Normal and Hyperglycemic Conditions
Marina G. Gobbo,1 Nishtman Dizeyi,2 Per-Anders Abrahamsson,2
Per-Anders Bertilsson,2 Viviane Sanches Masitéli,3 Eloisa Zanin Pytlowanciv,1
Sebastião R. Taboga,3 and Rejane M. Góes3
1
Department of Cell Biology, Institute of Biology, UNICAMP, Avenue Bertrand Russel, 6109 Campinas, SP, Brazil
Department of Clinical Sciences, Division of Urological Research, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden
3
Department of Biology, Institute of Biosciences, Humanities and Exact Sciences, UNESP, São José do Rio Preto, SP, Brazil
2
Correspondence should be addressed to Rejane M. Góes; [email protected]
Received 13 May 2015; Revised 22 June 2015; Accepted 24 June 2015
Academic Editor: Hiroshi Okamoto
Copyright © 2015 Marina G. Gobbo et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
The antitumor properties of melatonin (MLT) are known for prostate cancer cells. This study investigated whether MLT affects
prostate maturation and interferes with tissue injuries induced by diabetes. MLT was administered to Wistar rats from 5 weeks
of age in the drinking water (10 𝜇g/kg b.w.), and diabetes was induced at the 13th week by streptozotocin (4.5 mg/100g b.w., i.p.).
The animals were euthanized in the 14th and 21st weeks. MLT reduced the immunostained cells for androgen receptor (AR) by
10% in younger rats. Diabetes decreased cell proliferation and increased apoptosis. MLT treatment impeded apoptosis (𝑝 = 0.02)
and augmented proliferation (𝑝 = 0.0008) and PCNA content in prostate following long-term diabetes due to restoration of
testosterone levels and expression of melatonin receptor type 1B. The effect of MLT (500 𝜇M, 5 mM, and 10 mM) on androgendependent (22Rv1) and androgen-independent (PC3) cancer cells and human prostate epithelial cells (PNTA1) under normal and
hyperglycemic conditions (HG, 450 mg/dL) was analyzed. Contrary to PNTA1 and 22Rv1 cells, MLT improved the proliferation of
PC3 cells in hyperglycemic medium. The combined data indicated that MLT had proliferative and antiapoptotic effects in prostate
cells subjected to HG levels and it seems to involve specific MLT pathways rather than AR.
1. Introduction
Melatonin (MLT) is an indoleamine produced by the pineal
gland that controls the circadian cycle and acts as a neuromodulator, cytokine, and biological response modifier [1].
This hormone is widely known as an adjustor of the reproductive physiology to the environmental light in seasonally
dependent mammals [2]. MLT also has antioxidant [3–6]
and anti-inflammatory properties [7, 8]. The cellular action
of MLT is mediated by three receptor subtypes (MT1, MT2,
and MT3) coupled to G proteins. MLT signaling may also be
modulated by the activation of a series of nuclear receptors
referred to as retinoid Z receptors 𝛼 and 𝛽 [9, 10]. Depending
on the concentration, MLT can act directly in the cytosol
through calmodulin and quinone reductase 2 [11–13].
Data from Gilad et al. [14] indicated the presence of MLT
receptors in the rat prostate. The inhibition of xenografted
prostate tumor growth by MLT treatment has been reported
in rodents, and the antiproliferative action of MLT occurred
via activation of the MT1 receptor with consequent attenuation of calcium influx induced by sex steroids [15–17]. The
antitumor action of MLT in prostate cancer cell lines has
been attributed to changes in cell cycle, androgen receptor
(AR) translocation, and inhibition of angiogenesis through
reduced expression of factors that act under hypoxic conditions, such as hypoxia-inducible factor 1𝛼 (HIF-1𝛼) [18–25].
2
MLT also exerts a proapoptotic effect in tumor cells via p38
and c-jun terminal kinases (JNK); however, this effect is
independent of extracellular signal-regulated kinase (ERK)
activation [26]. This hormone has been associated with
increased sensitivity to some chemotherapeutic drugs [27].
Moreover, androgen-independent cell lines, such as PC3 and
DU145, are less sensitive to MLT compared to the androgendependent cells, such as LNCaP, CW22Rv1, and RWPE-1 [28–
30].
Because MLT has an antigonadotrophic effect in humans
and rodents by inhibiting testosterone synthesis in the testis
[31, 32], it is difficult to discriminate the direct influence of
this hormone in androgen-dependent organs, such as the
prostate. Few studies have considered the effects of MLT on
prostate in vivo, especially during sexual maturation.
The effects of diabetes on prostate histophysiology have
been investigated in rodents [33–42]. The alterations include
atrophy, impaired secretory activity, reduced cell proliferation, an increased number of apoptotic and epithelial basal
cells, extracellular matrix remodeling, impaired androgen
sensitivity, and phenotypic changes in stromal cells [34, 36,
38, 40, 42–48]. The prostatic response to diabetes has been
related to the reduction of serum testosterone levels and lack
of insulin, typical of this metabolic disorder [48, 49].
The diabetic condition is associated with a large production of reactive oxygen species resulting from hyperglycemia. The effectiveness of therapy with different antioxidants against oxidative stress caused by diabetes has been
extensively investigated experimentally [50–55]. Treatment
with ascorbic acid normalized the increased glutathione Stransferase (GST) activity and reduced epithelial apoptosis
in the prostate after one month of experimental diabetes
induced by streptozotocin [53]. MLT is a potent antioxidant,
and its synthesis is impaired under hyperglycemia [56]; thus,
it is worthwhile to investigate the efficacy of MLT treatment
under diabetic conditions and its influence on prostate cells
under hyperglycemia.
This study examined whether pre- and cotreatment with
MLT interferes with tissue damage induced in the prostate
of Wistar rats by experimental diabetes, particularly in terms
of proliferative activity, apoptosis, and AR expression. In this
study we report for the first time the effects of continuous
use of this neurohormone during sexual maturation on these
processes and prostate histology. We also compared the
influence of MLT under hyperglycemic condition on the proliferation and apoptosis of androgen-dependent (CW22Rv1)
and androgen-independent (PC3) cancer cells and human
prostate epithelial cells (PNTA1).
2. Materials and Methods
The rats were kept in polyethylene cages with wood shaving
substrate, subjected to light cycles (14 h of light and 10 h of
darkness) and a temperature of approximately 25∘ C. Food
(Presence, In vivo, Paulinia, SP, Brazil) and filtered water were
provided ad libitum.
After one week of adaptation, the animals were weighed
and randomly distributed into two experiments, with eight
groups total (10 animals per group). The short-term experiment consisted of a control group (C1), a control treated
with melatonin (M1), a one-week diabetic group (D1), and a
one-week diabetic group treated with melatonin (MD1). The
long-term experiment consisted of a control group (C2), a
control treated with melatonin (M2), an eight-week diabetic
group (D2), and an eight-week diabetic group treated with
melatonin (MD2).
MLT administration (Sigma Chemical Co., St. Louis, MO,
USA) was based on the procedures established by WoldenHanson et al. [57]. MLT was dissolved in ethanol and stored
in aliquots at −80∘ C. Such conditions were standardized by
application of various consumption preference and aversion
tests, and MLT did not affect the amount of water consumed
by rats. MLT was available to animals (groups M1, M2,
MD1, and MD2) in drinking water (10 𝜇g/kg body weight
in ethanol 0.001%/day) from 5 to 14 weeks old. The MLT
intake per day of this investigation was based on an average
daily water consumption of 80 mL/day/animal and an average
body weight of 350 g. Water bottles were protected from light
because MLT is a photosensitive molecule, and the liquid
content was changed every day.
Diabetes was induced on the 8th week of MLT treatment
(13 weeks old) in groups D1, D2, MD1, and MD2 by the
intraperitoneal injection of 4.5 mg/100 g of body weight of
streptozotocin (Sigma Chemical Co., Louis, MO, USA),
diluted in 1 mL of 0.01 citrate buffer. This solution was
injected after 24 h of fasting and anesthesia with ketamine and
xylazine (0.1 mL/100 g of body weight). Control animals were
injected with citrate buffer only.
Glucose levels were assessed two days after streptozotocin
injection through the glucose monitor Accu-Chek (Roche
Diagnostics, Mannheim, Germany) in the fingertips of the
paws. Animals with glucose levels above 200 mg/dL were
used in this study. Due to the higher water intake of diabetic
animals, the MLT dose was corrected for groups D1, D2, MD1,
and MD2 after the diagnosis of diabetes. Groups C1, M1, D1,
and MD1 were euthanized at 14 weeks old, whereas groups
C2, M2, D2, and MD2 were euthanized at 21 weeks old. Thus,
there was a short administration of MLT for groups M1 and
MD1 (9 weeks) and a prolonged treatment for groups M2
and MD2 (16 weeks). The rats were euthanized using CO2
inhalation and then decapitated for blood collection.
2.1. In Vivo Experimental Design. Male Wistar rats (𝑛 = 80)
were obtained from the breeding house of São Paulo State
University (Botucatu, SP, Brazil) in the 5th week of life (weaning). This experiment was conducted according to the ethical
principles adopted by the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes,
and all procedures were approved by the Ethics Committee
on Animal Use of IBILCE/UNESP (Protocol 051/2011 CEUA).
2.1.1. Hormone Dosages. The blood samples were collected
after decapitation and plasma was separated through centrifugation at 1,200 g and frozen at −80∘ C for analysis of
testosterone levels. The measurements were performed by a
capture/sandwich ELISA (antibody-antigen-antibody) using
specific commercial kits (R&D Systems, Inc., Minneapolis,
MN, USA), with a sensitivity of 14.0 pg/mL and a variation for
interassays of 11.3 pg/mL. The readings were performed using
an Epoch Multi-Volume Spectrophotometer System (BioTek
Instruments, VT, USA).
2.1.2. Light Microscopy. Ventral prostates were removed and
weighed. Fragments of ventral prostate were fixed by immersion in 4% formaldehyde freshly prepared in phosphate buffer
pH 7.2 and methacarn (1 : 3 : 6 of acetic acid, chloroform, and
methanol) and embedded in Paraplast. The histological sections stained with hematoxylin-eosin were used for general
morphological studies and immunocytochemical analysis.
The sections were observed under a bright-field microscope
(Olympus CH30) coupled with a charge-coupled camera. The
digitization of selected microscopic fields and quantitative
analyses were performed using an image analysis system
(Image-Pro Plus Media Cybernetics, version 6.0 for Windows
software, Bethesda, MD, USA).
2.1.3. Immunohistochemistry. Immunocytochemical staining
for proliferating cell nuclear antigen (PCNA), androgen
receptor (AR), and melatonin receptor type 1B (MTR1B)
was assessed using specific antibodies purchased from Santa
Cruz Biotechnology (AR and PCNA, Santa Cruz, CA, USA)
and Novus Biologicals (MTR1B, Novus Biologicals, Littleton,
CO, USA). Sections were subjected to antigen retrieval in
citrate buffer (10 mM, pH 6) for 20–40 min, followed by
the blocking of endogenous peroxidase with 3% H2 O2 in
methanol or water (for MTR1B), and immersed at 3% normal
bovine serum (for PCNA and MTR1B) or 5% dry milk (for
AR) in PBS for 1 h to block nonspecific protein. After PBS
washing, incubation with primary antibodies diluted in 1%
BSA was carried out: AR (sc816, rabbit polyclonal, 1 : 100,
overnight at 4∘ C), PCNA (sc56, mouse monoclonal, 1 : 100,
1 h at 37∘ C), and MTR1B (NLS932, rabbit polyclonal, 1 : 75,
overnight at 4∘ C). Then, the tissue sections used for PCNA
and AR were incubated at RT with a Polymer/peroxidase kit
(Novolink Polymer, Novocastra, Norwell, MA, USA) for 1 h.
The tissue sections used for MTR1B were incubated at 37∘ C
with secondary antibody and then with avidin/biotin (ABC
Staining Systems, Santa Cruz Biotechnology, CA, USA) for
45 min.
The reaction was detected with 0.1% diaminobenzidine
(DAB) and 0.02% H2 O2 in PBS, and the sections were
counterstained with hematoxylin. Five animals per group and
3 prostatic fragments per animal were used to quantify the
proliferation levels, AR-positive cells, and MTR1B positive
areas. The sections were digitalized, and 30 contiguous fields
were observed in the 40X objective. AR and PCNA-positive
cells were quantified by counting the number of positive
nuclei and dividing it by the total number of nuclei per
visual field. Areas showing specific staining for MTR1B were
evaluated using a 130-point reticulum and the marked area
was counted as previously done [45]. Data were expressed as
relative frequency (%).
2.1.4. Detection of Apoptotic Cells. Apoptotic cells were
detected in situ using the DNA fragmentation assay associated with cell death based on a Terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) reaction (TdTFragel-Calbiochem, CN Biosciences, La Jolla, CA, USA)
3
following the manufacturer’s instructions. The negative controls were obtained by omitting the incubation with TdT
enzyme, and the slides were stained with hematoxylin. The
quantification of apoptotic cells was performed in the same
manner as the immunohistochemistry reaction for PCNA.
2.1.5. Western Blotting Analysis. The PCNA protein content
in the prostate samples was quantified by Western blotting.
Prostate samples were homogenized at 4∘ C in cell lysis buffer
(20 mM Tris-HCl, 150 mM NaCl, 1% Triton X100, 2% SDS)
containing 100 mM phenylmethylsulfonyl fluoride, 100 mM
sodium orthovanadate, and a protease inhibitor cocktail
(1 : 1,000, Sigma, St. Louis, MO, USA Chemical Co.). Lysates
were centrifuged at 13,000 g at 4∘ C for 15 min, the supernatants were collected, and the protein concentration was
determined using the Bradford method [58]. Laemmli sample buffer with 5% 𝛽-mercaptoethanol was added to equal
amounts of protein (150 𝜇g). The samples were then separated
by SDS-PAGE on 12% polyacrylamide Tris-glycine gels and
electroblotted to nitrocellulose membrane (GE Healthcare)
using Bio-Rad assay equipment (Bio-Rad, Hercules, CA,
USA). Nonspecific proteins were blocked with 5% nonfat dry
milk in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2%
Tween-20) for 1 h at room temperature, and the membranes
were probed with primary antibody, PCNA (1 : 300) overnight
at 4∘ C in 1% BSA in TBST. Then, the membranes were washed
in TBST and incubated for 1 h at 4∘ C with the specific secondary horseradish peroxidase-conjugate antibody (1 : 200 in
1% BSA in TBST), followed by 3 washes in TBST. Antibody
immunolabeling was revealed by an ECL chemiluminescent
detection kit (Healthcare). 𝛽-actin was used as a control for
protein expression. Densitometric analysis was performed
using the Image J 1.34 software (Wayne Rasband, Research
Services Branch, National Institute of Health, Bethesda, MD,
USA).
2.2. In Vitro Experimental Design. Three human cell lines
were used in this study: a nontumoral cell line (PNTA1) and
two prostate cancer cell lines (CW22Rv1, herein referred to as
22Rv1 and PC3). 22Rv1 (CRL-2505, from xenograft line) and
PC3 (CRL-1435, from bone metastasis) were purchased from
the American Type Culture Collection (ATCC, Manassas,
VA, USA). PNTA1 cells (#95012614) were obtained from the
Health Protection Agency (UK). The cells were grown in
RPM1640 medium containing 10% fetal bovine serum (FBS)
and 1% penicillin-streptomycin (Life Technologies, Paisley,
UK) in a humidified atmosphere of 95% air and 5% CO2
at 37∘ C. Cells were fed every 2-3 days and subcultured
once they reached 70–80% confluence. The first phase of
the investigation consisted of dose-related experiments with
different melatonin concentrations (Sigma Chemical Co.,
St. Louis, MO, USA). Melatonin (MLT) was always freshly
prepared in a 40% DMSO stock solution (Sigma Chemical
Co., St. Louis, MO, USA) and diluted to different concentrations in the culture medium. After determining the correct
range of MLT doses, the cell lines were preincubated in
hyperglycemic medium with 450 mg/dL of glucose (DMEM;
Invitrogen, Carlsbad, CA, USA) and then treated with MLT.
Two different culture media were used because the 22Rv1
4
cells seeded in DMEM failed to attach properly. Thus, for the
experiments with high glucose (HG) conditions, cells were
cultured first with RPMI and then with DMEM with high
glucose levels. The initial analysis of MLT effects using the
light microscope revealed that 22Rv1 cells were more sensitive
to indoleamine than the other cell types because more cells
became detached when they were treated with 10 mM of MLT.
Thus, a reduced MLT concentration (5 mM) was used for
22Rv1 cells, whereas PNTA1 and PC3 cells received 10 mM
MLT. With this correction, a proportional cell density was
maintained, which is crucial for the assays in this study.
Thus, the following conditions were evaluated: in normal
conditions (NC), the cells were plated with RPM1 medium
and incubated with DMSO (at the same concentration of the
highest dose of melatonin used). Melatonin treatment was
performed in the following concentrations: 500 𝜇M, 5 mM,
and 10 mM. Exposure to HG conditions was performed by
replacing the RPM1 medium with hyperglycosylated DMEM
(450 mg/dL) and incubating with the vehicle (DMSO). The
concomitant exposure to HG and MLT was performed in the
same manner as described separately above.
2.2.1. Proliferation Assay. For this assay, 96-well flatbottomed plates were used at a density of 3,000 cells/well
with 100 𝜇L of medium. The cells were preincubated with
HG DMEM for 1 day for the short-term experiment and
7 days for the long-term experiment. Then, the cells were
treated with DMSO and MLT (500 𝜇M, 5 mM, or 10 mM)
for an additional 1 and 2 days. The 3-(4,5-dimethylthiazol2yl)-2,5-diphenyl tetrazolium bromide (MTT) is reduced
by metabolically active cells, which results in the formation
of purple formazan. A solution of 50% MTT in PBS was
applied to each well, and the plate was incubated for 2 h
in a humidified incubator at 37∘ C in an atmosphere of 5%
CO2 protected from light. 100 𝜇L of isopropanol with 0.04 N
HCl was added to each well and mixed by tapping gently
on the plate. After 15 min, the absorbance was measured
on an ELISA plate reader with a test wavelength of 570 nm
and a reference wavelength of 630 nm. The results were
expressed in absorbance values as a mean ± S.D. of the two
experiments.
2.2.2. Flow Cytometry. Cell cycle distribution (G0/G1, S,
and G2/M) and apoptosis were analyzed by flow cytometry
based on DNA content. 22Rv1, PNTA1, and PC3 cells were
seeded in 6-well plates with a density of 100,000 cells and
1,000 𝜇L of RPM1. Some of the wells were preincubated with
hyperglycemic DMEM for 24 h and then treated with MLT
for an additional 24 h. The cells were washed in phosphatebuffered saline (PBS) without Ca2+ or Mg2+ , followed by centrifugation (400 g, 5 min, 4∘ C). The fixation was performed
in ice cold 70% ethanol followed by vortexing. The samples
were stored at 4∘ C for 2 h and centrifuged again (400 g, 5 min,
4∘ C). For staining with propidium iodide (PI), cells were
washed with cold PBS, centrifuged (300 g, 5 min, 4∘ C), and
then suspended in a 500 𝜇g/mL PI solution (#11348639001;
Roche, Mannheim, Germany) protected from the light at
room temperature for 40 min. The samples were transferred
to glass tubes (5 mL Falcon tubes) and kept on ice. The cells
were gated, and the DNA content of at least 4,000 labeled
cells was quantified with a FACSCalibur instrument (BD
Bioscience, San Jose, CA, USA). During data analysis, doublet
discrimination was performed using a two-parameter dot
plot of FL2-Area versus FL2-Width. Data presented were
analyzed using FlowJo 10.0.7 software (Treestar, Inc., Ashland, OR, USA). The percentage of fragmented DNA was
considered an indirect measure of apoptosis.
2.2.3. Statistical Analyses. Data were tested considering the
assumptions of normality and homogeneity of variances
according to the Shapiro-Wilk and Levene tests, respectively.
The groups that have made such assumptions (parametric
data) were compared by applying a t-test or one-way analysis
of variance (ANOVA) followed by Tukey’s test (post hoc).
Data that did not fit these assumptions (nonparametric
data) were compared by applying the Mann-Whitney U
or Kruskal-Wallis test followed by Dunn’s test (post hoc).
Statistical analyses were performed between groups of the
same experimental period and between different periods
for the same treatment (for biometric data only) using the
Statistica 9.0 software (Statsoft Inc., Tulsa, OK, USA). Data
were expressed as a mean ± standard deviation, and 𝑝 < 0.05
was considered statistically significant.
3. Results
3.1. In Vivo Experiment
3.1.1. Physiological and Biometric Parameters. Body weight
gain (Figure 1(a)) did not vary among short-term experimental groups, except for a diminution in group MD1. Both
diabetic groups from the longer duration experiment had
smaller body weight gain, independently of MLT treatment
(𝑝 < 0.0001). Prostate weight was not affected by MLT
treatment under healthy conditions as well as in diabetes conditions in short- and long-term experiments (Figure 1(b)).
MLT administration prevented partially prostate atrophy
caused by short-term diabetes (𝑝 = 0.0025, Figure 1(b)),
whereas this hormone was not able to prevent atrophy of the
gland in two-month-old diabetic animals (𝑝 < 0.01). Ninetyone percent of the animals exhibited blood glucose levels
of 404 mg/dL, as shown in Figure 1(d). Glycemia decreased
by 18% from C1 to C2 (𝑝 = 0.026), and MLT did not
change this parameter (Figure 1(d)). However, MLT affected
the testosterone synthesis of healthy animals (Figure 1(e)) in
both short- (decreased by 24%) and long-term (decreased
by 34%) experiments. The serum androgen levels were also
drastically decreased by induced diabetes (𝑝 < 0.005). Group
MD2 exhibited higher levels of this hormone compared to
group D2 (Figure 1(e)).
3.1.2. Expression of the Androgen Receptor Using Immunocytochemistry. The treatment of healthy rats with MLT from
weaning to 9 weeks reduced the number of AR-positive cells
in the prostatic epithelium at early adulthood (𝑝 = 0.03;
Figures 2(c) and 2(i)). However, the frequency of AR-positive
cells did not change when the treatment was extended for
an additional 7 weeks (Figures 2(d) and 2(i)). One week
5
A∗
a, b
300
Prostate weight
800
Body weight gain
400
A∗ A∗
600
A∗
a
200
(mg)
(g)
a
a, b
a
a, b
B
b
400
B
B
b
B∗
200
100
0
M1
C1
C2
D1 MD1
M2
0
D2 MD2
C1 M1 D1 MD1
(a)
M2 D2 MD2
(b)
Prostate relative weight
2
C2
Glycemia
600
B
B
(103 )
a
a
b
A∗ A∗
a
a
A∗
A
1
200
0.5
0
C1 M1 D1 MD1
C2
b
400
(mg/dL)
1.5
0
M2 D2 MD2
a
C1
a
M1
D1 MD1
(c)
A∗
A∗
C2
M2
D2 MD2
(d)
Serum testosterone
800
A∗
600
B∗
(ng/dL)
B∗
400
a
C∗
c
200
0
C1
b, c
c
M1
D1
MD1
C2
M2
D2
MD2
(e)
Figure 1: Mean and standard deviation of body weight gain (a), prostate wet weight (b), prostate relative weight (c), blood glucose levels
(d), and serum testosterone (e). C1: short-term control; M1: short-term control treated with MLT; D1: short-term untreated diabetic; MD1:
short-term diabetic treated with MLT; C2: long-term control; M2: long-term control treated with MLT; D2: long-term untreated diabetic; and
MD2: long-term diabetic treated with MLT. Light bars: short-term experiment and dark bars: long-term experiment (𝑁 = 10 animals/group).
Different lowercase letters indicate significant differences between experimental groups C1, M1, D1, and MD1 (parametric data: prostate
weight, relative prostate weight, and serum testosterone; nonparametric data: body weight gain, glycemia), and different capital letters indicate
significant differences between groups C2, M2, D2, and MD2 (parametric data: body weight gain, prostate weight, relative prostate weight,
and serum testosterone; nonparametric data: glycemia). ∗ Significant difference between experimental periods (t-test or Mann-Whitney U
test).
6
l
s
e
l
e
s
(C1)
(C2)
(a)
(b)
s
s
l
l
e
e
(M1)
(M2)
(c)
(d)
s
e
e
s
l
l
(D1)
(D2)
(e)
(f)
e
l
s
e
l
s
(MD2)
(MD1)
(g)
(h)
Figure 2: Continued.
7
100
B
80
a
b
A, B
A
60
(%)
A
c
c
D1
MD1
40
20
0
C1
M1
C2
M2
D2
MD2
(i)
Figure 2: Immunohistochemistry ((a)–(h)) for the androgen receptor and quantification of AR-positive cells (i) in the acinar epithelium of rat
ventral prostate (brown nuclei). (a) Short-term control (C1); (b) short-term control treated with MLT (M1); (c) short-term untreated diabetic
(D1); (d) short-term diabetic treated with MLT (MD1); (e) long-term control (C2); (f) long-term control treated with MLT (M2); (g) longterm untreated diabetic (D2); (h) long-term diabetic treated with MLT (MD2). e: epithelium; l: lumen; s: stroma; arrow: AR-positive cells.
Magnification: 400x, bar = 25 𝜇m. (i) AR-positive epithelial cells frequency (%); light bars: short-term experiment and dark bars: long-term
experiment (𝑁 = 5 animals/group). Different lowercase letters indicate significant differences between experimental groups C1, M1, D1, and
MD1 (parametric data), and different capital letters indicate significant differences between groups C2, M2, D2, and MD2 (nonparametric
data).
of diabetes decreased the AR immunostained cells by 40%
compared to group C1 (Figures 2(e), 2(g), and 2(i)), and
MLT treatment did not prevent this depletion (𝑝 < 0.0001).
Following two months of diabetes, the proportion of cells
expressing AR nearly doubled (40%), and MLT consumption
abrogated this increase (Figures 2(f), 2(h), and 2(i)).
The relative frequency of stained areas was increased by ∼30%
due to melatonin administration to healthy rats and to experimental diabetes (Figures 5(c)–5(f) and 5(k)). MLT avoided
such augmentation of relative frequency of MTR1B stained
areas in diabetic animals of both experiments (Figures 5(g),
5(h), and 5(k)).
3.1.3. Proliferation, Apoptotic Rates, and PCNA Content. The
frequency of cell proliferation did not alter in the prostates of
groups M1 and M2 (Figures 3(a), 3(b), 3(c), 3(d), and 3(i)).
Cell proliferation in the gland was reduced by 80% after one
week and by 40% after two months of diabetes (Figures 3(e),
3(f), and 3(i)). MLT therapy prevented the decrease in cell
proliferation in the prostate caused by long-term, but not
short-term diabetes (Figures 3(g), 3(h), and 3(i)). Western
blotting analysis showed no variation in PCNA expression
in the short-term experiment; however, higher expression of
PCNA was detected (60%) for group MD2 compared to group
D2 in the long-term experiment (Figure 3(j)).
The administration of MLT did not affect apoptosis in the
prostate of healthy rats in both experiments, although there
was a nonsignificant augmentation in group M2 (Figures
4(a), 4(b), 4(c), 4(d), and 4(i)). The number of apoptotic
cells increased by two-fold in the gland of diabetic animals
(Figures 4(e), 4(g), and 4(i)). MLT ameliorated the levels of
apoptosis in the prostate of rats after two months of diabetes
(Figures 4(h) and 4(i)).
3.2. In Vitro Results
3.1.4. Immunolocalization and Quantification of MTR1B. The
immunolocalization of melatonin receptor type 1B occurred
specially in the prostate epithelium (Figure 5). Tissue sections
of rat brain were used as a positive control and the neuron cell
bodies presented positive staining for MTR1B (Figure 5(i)).
3.2.1. Effects of MLT and HG Medium on Cell Proliferation.
PNTA1 cells were not affected by MLT under NC after 24 or
48 h of incubation (Figure 6(a)). The short incubation with
HG medium and concomitant treatment with MLT at 10 mM
decreased the amount of mitosis (𝑝 = 0.02; Figure 6(b)).
This effect was not observed in the 7-day preincubation
experiment (Figure 6(c)).
Cancer cell line 22Rv1 exhibited a decrease in cell proliferation (𝑝 = 0.008) after 1 day of exposure to 5 mM MLT
under NC (Figure 6(d)). The same exposure period to MLT,
but with a previous incubation of one day in HG medium,
provoked the same effect, decreasing the cell proliferation by
23% with 5 mM (Figure 6(e)). These cells exhibited an early
increase in proliferation after a short preincubation with the
HG condition (Figure 6(e)) and an inverse pattern after a long
preincubation, decreasing the viability to approximately 40%,
independently with MLT treatment in both time intervals
(Figure 6(f)).
Exposure to the lowest MLT concentration under NC for
24 h favored PC3 cell proliferation. However, after 48 h, MLT
at the highest molarity diminished PC3 viable cells by 60%
(Figure 6(g)). The preincubation of PC3 cells in HG medium
for one day had a synergistic action associated with MLT on
the proliferation index after 24 h in a dose-dependent manner
8
e
s
e
l
e
s
l
(C1)
(C2)
(a)
(b)
l
l
s
s
(M1)
e
(M2)
e
(c)
(d)
l
s
e
l
e
(D2) s
(D1)
(e)
(f)
l
e
s
s
l
e
(MD1)
(MD2)
(g)
(h)
9
-PCNA-34 kDa-
0.5
4
0.4
a
a
(%)
3
2
A
A
A, B
1
0
C1
M1
B
b
b
D1
MD1
C2
M2
D2
Relative to 𝛽-actin
5
-𝛽-actin-42 kDa-
A, C
a
0.3
a
a
C
A
a
0.2
B
0.1
MD2
0
C1
M1
D1 MD1
(i)
C2
M2
D2 MD2
(j)
Figure 3: Proliferation index, evaluated by immunocytochemistry for proliferating cell nuclear antigen (PCNA) and Western blotting. (a)
Short-term control (C1); (b) short-term control treated with MLT (M1); (c) short-term untreated diabetic (D1); (d) short-term diabetic treated
with MLT (MD1); (e) long-term control (C2); (f) long-term control treated with MLT (M2); (g) long-term untreated diabetic (D2); (h)
long-term diabetic treated with MLT (MD2). e: epithelium; l: lumen; s: stroma; arrow: PCNA-positive cells (brown nuclei). Magnification:
400x, bar = 25 𝜇m. (i) Relative frequency (%) of PCNA-positive cells. (j) Western blotting for PCNA in ventral prostate extracts and the
respective densitometry analysis. 𝛽-actin was used as internal control loading. Light bars: short-term experiment and dark bars: long-term
experiment (𝑁 = 5 animals/group). Different lowercase letters indicate significant differences between experimental groups C1, M1, D1, and
MD1 (parametric data: PCNA protein content; nonparametric data: frequency of PCNA-positive cells), and different capital letters indicate
significant differences between groups C2, M2, D2, and MD2 (parametric data: PCNA protein content; nonparametric data: frequency of
PCNA-positive cells).
(Figure 6(h)). This effect was enhanced 48 h after the addition
of MLT, mainly at the 10 mM concentration. One week of
previous exposure to HG augmented PC3 cell proliferation,
compared to 1 day of previous incubation, regardless of the
treatment with neurohormone, especially after 2 days of
10 mM MLT exposure (Figure 6(i)).
3.2.2. Cell Cycle Analysis by Flow Cytometry. The 1-day
exposure of PNTA1 cells to 10 mM MLT under NC increased
the population of G2/M cells by 36% in comparison to control
medium (Figures 7(a) and 7(b)). The incubation of these cells
with HG for 24 h increased the G2/M cell population by 52%
(Figure 7(c)), and this increase was higher (92%) following
24 h of MLT incubation (Figure 7(d)). However, there was a
90% increase in the number of cells with DNA fragmentation
when this cell line was incubated in HG medium and a
105% increase when incubated in HG medium and MLT
(Figure 7(d)). There was a decrease in cell population at the
S phase in both groups incubated with HG and a reduction
in the G0/G1 cell population after HG and MLT incubation
(Figure 7(d)).
Melatonin treatment of 22Rv1 cells cultivated in NC
for 24 h increased the percentage of apoptosis by 30%, the
number of cells in interphase by 28%, and the number
of G2/M phase cells by 64% (Figures 7(e) and 7(f)). The
exposure of 22Rv1 cells to HG medium also increased the
apoptotic (36%) and G2/M (34%) cells (Figures 7(e) and
7(g)), and the addition of MLT attenuated these alterations
(Figure 7(h)).
In the PC3 cell line, MLT caused a decrease of 30%
in cells in the G0/G1 phase (Figures 7(i) and 7(j)) and an
augmentation of 92% in G2/M cells. HG conditions did not
affect the proliferation (G2/M) of this lineage but increased
the number of apoptotic cells by 90% (Figure 7(k)). Such
alterations were restored by MLT (Figure 7(l)).
4. Discussion
In addition to corroborate with previous evidence that
diabetes markedly reduces cell proliferation and increases
apoptosis in the prostate [37, 39, 43, 53], the present results
indicate, for the first time, that MLT administration (10 𝜇g/kg
body weight/day) to Wistar rats retrieved the proliferative
activity of the prostate after two months of experimental
diabetes. This retrieval was associated with improvement of
the androgen synthesis that is known to be impaired in
chronic diabetes [43, 45, 53]. In addition it was observed
that prostate response to MLT treatment differed during
the disease progression, following alterations of testosterone
levels, because modifications of proliferation and apoptosis in
the gland or improvement of circulating testosterone was not
found in the animals after short-term diabetes. We also noted
that MLT administration to healthy rats since weaning had a
negative action on testosterone synthesis but did not interfere
with proliferative and apoptotic response in prostate. This
suppressive action of MLT on androgen levels of healthy
rats was expected and it may be due to the modulation of
gonadotropin-inhibitory hormone (GnIH) and its respective
receptor [59], the decrease in LH levels [32], or the direct
action of MLT in interstitial testicular cells through melatonin receptors [60]. Presumably, no effect was verified on cell
proliferation and apoptosis rates because testosterone reduction did not induce a conspicuous change in AR expression
in the gland of MLT-treated healthy rats, as indicated by
10
l
s
l
s
e
e
(C2)
(C1)
(a)
(b)
l
e
e
l
s
s
(M1)
(M2)
(c)
(d)
s
e
l
e
l
s
(D2)
(D1)
(e)
(f)
l
s
e
e
s
(MD1)
l
(MD2)
(g)
(h)
11
2
(%)
1.5
b
1
a
0.5
0
C1
A, B B, C
b
A, C
a
M1
A
D1 MD1
C2
M2
D2 MD2
(i)
Figure 4: Apoptotic cells quantified by the TUNEL method (brown nuclei). (a) Short-term control (C1); (b) short-term control treated
with MLT (M1); (c) short-term untreated diabetic (D1); (d) short-term diabetic treated with MLT (MD1); (e) long-term control (C2); (f)
long-term control treated with MLT (M2); (g) long-term untreated diabetic (D2); (h) long-term diabetic treated with MLT (MD2). e:
epithelium; l: lumen; s: stroma; arrow: PCNA-positive cells (brown nuclei). Magnification: 400x, bar = 25 𝜇m. (i) Relative frequency of
apoptotic cells (%). Light bars; short-term experiment and dark bars: long-term experiment (𝑁 = 5 animals/group). Different lowercase
letters indicate significant differences between experimental groups C1, M1, D1, and MD1 (nonparametric data), and different capital letters
indicate significant differences between groups C2, M2, D2, and MD2 (nonparametric data), according to ANOVA followed by the Tukey
(post hoc) or Kruskal-Wallis test followed by Dunn’s test (post hoc).
a slight decrease of 10% in the frequency of AR-positive cells
in short-term experiment. Then, our data indicated that, even
at lower doses than usually employed previously [6, 61, 62],
the proliferative and antiapoptotic effects of MLT treatment
in the prostate of animals with chronic diabetes are likely
due to its indirect action on circulating testosterone levels
and also reflected the improvement of androgenic action in
the gland under diabetes as demonstrated by AR-positive
cells frequency. The increase of serum testosterone levels in
diabetic animals treated with melatonin should be analyzed
carefully, especially when melatonin is intended to be applied
as a coadjutant in the treatment of prostate cancer for patients
with metabolic disturbances, such as diabetes.
Furthermore, testosterone influence in the prostate
response to MLT in diabetes was also triggered by conventional membrane receptor. MTR1 had been related to
antiproliferative response of prostate cancer cells to MLT,
which leads to the downregulation of activated AR signaling and upregulation of p27kipi in 22Rv1 cells [63]. Our
immunohistochemical data showed a significant reduction
of melatonin receptor type 1B (MTR1B) for MD2 group
in relation to D2 group, which can support the increased
proliferation index found in these animals. In addition, in a
parallel investigation with a similar experimental protocol,
we demonstrated that MLT had effective antioxidant action
in this gland under diabetes by equilibrating glutathione Stransferase (GST), catalase, and glutathione peroxidase (GPx)
activities [64]. The MLT ability to attenuate the production
of reactive species (ROS) and also regulating the expression
of proteins of the apoptotic pathways, such as Bcl-2 and Bax
[65, 66], have been related to the antiapoptotic property of
this neurohormone. The increase in PCNA content in the
melatonin-treated diabetic group (MD2), besides expressing
an increase in cell proliferation, can also corroborate the
protective action of MLT because PCNA is also involved
in DNA damage repair and epigenomic maintenance [67].
Taken together the in vivo results demonstrate that response
of diabetic prostate to the MLT treatment was mainly due to
the alterations in androgen levels, as expected, but they also
suggest the involvement of MTR1B receptor. Most evidence
in the literature points to an antiproliferative action of MLT
on prostate cancer cells, and in vivo studies with normal cells
are scarce [14, 68, 69]. Therefore our results for the long-term
diabetic group treated with MLT are in contrast to in vitro
studies and they also indicate a differential action of MLT
during normoglycemia or hyperglycemia.
To better clarify the implications of MLT from its
secondary effect on androgen levels, and the interferences
of hyperglycemia and AR expression, we examined MLT
influence on cell proliferation of human prostate epithelium
cells and prostate cancer cell lineages. It is known that MLT
can detain mitosis through the attenuation of calcium influx
induced by dihydrotestosterone (DHT) and downregulation
of androgen signaling due to the nuclear exclusion of AR
[17, 21, 22]. The inhibition of tumor enlargement due to the
antiproliferative action of MLT is mediated by cell cycle delay
at the G0/G1 phase and a shorter duration in the S phase
by reducing the levels of cyclin D1 [16, 18, 19]. The usage of
both tumor and nontumor cell lines was pertinent because it
was observed that 8% of diabetic animals presented prostate
carcinoma and 15% presented high grade intraepithelial
neoplasia (data not shown). The glucose concentration used
in the medium (450 mg/dL) is equal to the glycemic index
of diabetic animals in the present investigation. Our study
was performed with high MLT concentrations (5 mM and
10 mM) compared with most previous studies, which utilized
doses in the nM and 𝜇M range [21, 24, 25, 63]. Higher MLT
concentrations were expected to be effective in concomitantly
covering three different cell lines. The uptake of MLT by
prostate cancer cells is poor and dependent on the cell
cycle phase, and the cells in the present investigation were
not synchronized. Therefore, high concentrations of MLT
12
s
l
l
s
l
l
e
e
(C1)
(C2)
(a)
(b)
l
l
e
s
e
e
s
l
(M2)
(M1)
(c)
(d)
s
e
l
e
l
(D1)
(D2)
(e)
(f)
s
l
s
e
e
l
l
e
e
*
l
(MD1)
(+)
(MD2)
(g)
(h)
(i)
13
20
b
18
16
e
l
b
a
b
c
14
b
12
(%)
s
l
10
a
a
8
6
4
2
0
(−)
C1
(j)
M1
D1 MD1
C2
M2
D2 MD2
(k)
Figure 5: Tissue localization of melatonin receptor type 1B (MTR1B) in the ventral prostate of rats. (a) Short-term control (C1); (b) short-term
control treated with MLT (M1); (c) short-term untreated diabetic (D1); (d) short-term diabetic treated with MLT (MD1); (e) long-term control
(C2); (f) long-term control treated with MLT (M2); (g) long-term untreated diabetic (D2); (h) long-term diabetic treated with MLT (MD2). (i)
Negative control; (j) positive control, brain tissue section of rat. (k) Relative frequency (%) for MTR1B-positive areas. e: epithelium; l: lumen;
s: stroma. Magnification: 400x, bar = 25 𝜇m. Light bars: short-term experiment and dark bars: long-term experiment (𝑁 = 5 animals/group).
Different lowercase letters indicate significant differences between experimental groups C1, M1, D1, and MD1 (nonparametric data), and
different capital letters indicate significant differences between groups C2, M2, D2, and MD2 (parametric data), according to ANOVA followed
by the Tukey (post hoc) or Kruskal-Wallis test followed by Dunn’s test (post hoc).
in the medium do not necessarily correlate with higher
internal concentrations [29]. Thus, the solubility of MLT was
hampered, and a higher concentration of DMSO had to be
used to dissolve it.
Proliferation assays did not indicate a drastic antiproliferative and proapoptotic action of MLT under NC, as
reported previously [70]. These attenuated unintended effects
may be due to the shorter incubation times employed here.
Sainz et al. [71] disposed the indoleamine for 2–6 days,
allowing more time for its metabolism. Hevia et al. [72]
proved that melatonin is still available in the medium and
not transformed into its metabolites by prostate cancer cells
until 24 h of incubation. Moreover, the intracellular levels of
melatonin decay after 24 and 48 h [29], which support the
possibility of differences in the influence of MLT between
short and continuous exposure.
PNTA1 are human nontumoral cells that were used here
as an analogous for the epithelium cells of rat ventral prostate.
As observed in vivo, MLT did not elicit an antiproliferative
response in PNTA1 cells under normal conditions and HG
medium increased the percentage of fragmented DNA. As
also observed in a previous research, there was no increase in
proliferation by the association of PNTA1 cells and DMEM
with HG (450 mg/dL) [73]. Androgen receptor positive but
not androgen sensitive cells, 22Rv1, were highly sensitive to
MLT in NC, particularly at high concentrations, but when
incubated in HG medium, the MLT was capable of reducing
cell proliferation of 22Rv1 after a short incubation period.
PC3 cells revealed a different behavior from the other cells
exhibiting a late antiproliferative response to MLT in NC and
a synergistic proliferative effect with high glucose medium,
as showed by both MTT and flow cytometry data. Thus, the
prolonged incubation with HG medium impaired the mitosis
of 22Rv1 and PNTA1 cells but improved this parameter for
PC3 cells. Hevia et al. [74], using LNCaP and PC3 cells incubated under HG concentrations, observed an antiproliferative
effect of MLT that was caused via modulation of glucose
transporter type 1 (GLUT1). Our results do not completely
agree with the above findings, particularly in the case of PC3
cells. These discrepancies can be explained by the different
incubation times and concentrations of HG.
Although the use of in vivo and in vitro experiments
allowed observing some parallelisms in the results, the
differences between the microenvironments in which these
cells are inserted should not be neglected. In this way, the
in vivo experiment has a much more complex panel of
variables. The prostate secretory epithelium is composed
of various cell types, interrelated with each other, as the
basal cells, transit-amplifying cells, intermediate cells, and
the luminal cells [75]. Thus, none of the cell lines used in
this research can represent the population heterogeneity of
prostatic epithelium. It is worthy to mention that short-term
diabetes drastically reduced the frequency of AR-positive
cells in the prostate and this effect probably persists at later
stages of the disease as previously reported by our research
group after one-month of streptozotocin-induced diabetes
[53]. Therefore the results observed for the longer duration
experiment reflect the MLT influence in cells whose majority
does not express AR. In this context, PC3 cells displayed
a comparable behavior to the prostatic cells under chronic
diabetes. Furthermore, in this case, the MLT response in vitro
can be mediated by other signaling pathways, such as those
related to specific membrane or nuclear receptors for this
hormone.
14
PNTA1
0.1
0
1
0.2
∗
0.1
0
2
1-day preincubation with HG
1
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
∗
0
2
1
0.4
0
2
1
PC3
∗
+
0.1
2
0.2
0.1
0
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
(f)
0.4
0.3
∗
∗∗
2
(day)
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
(g)
0.2
0.1
1
(day)
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
2
(day)
0.3
0.3
PC3
0.1
(e)
Normal condition
1
0.2
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
(d)
0
0.3
(day)
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
0.2
(c)
∗
(day)
0.4
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
0.2
0.1
2
(day)
22Rv1
22Rv1
22Rv1
0.3
0.2
1
1
(b)
Normal condition
0
0.1
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
(a)
0.1
0.2
0
2
(day)
(day)
0.3
0.3
PC3
PNTA1
0.2
0.3
PNTA1
Normal condition
0.3
(h)
0
1
2
(day)
DMSO
500 𝜇M
5 mM (22Rv1)/10 mM
(PC3 and PNTA1)
(i)
Figure 6: MTT assay (absorbance values) for cell proliferation for PNTA1 ((a), (b), and (c)), 22Rv1 ((d), (e), and (f)), and PC3 cells ((g),
(h), and (i)) under normal conditions ((a), (d), and (g)), 1 day of preincubation with hyperglycemic medium ((b), (e), and (h)), 7 days of
preincubation with hyperglycemic medium ((c), (f), and (i)), and treatment with DMSO, 500 𝜇M, 5 mM, or 10 mM of MLT for 1 and 2
days. ∗ represents significant differences among the different concentrations of melatonin exposure for 1 day (parametric data). + represents
significant differences among the different concentrations of melatonin exposure for 2 days (parametric data).
5. Conclusion
Our in vivo data indicated that under chronic diabetes
exogenous melatonin had a proliferative and antiapoptotic
effect due its indirect action on testosterone circulating levels
and also direct effect through specific MTR1B receptor. PC3
cell line showed a similar result to the in vivo experiments,
because an increase in mitosis was elicited at longer preincubation times with hyperglycemic medium and MLT. As
PC3 cells are androgen-independent prostate cancer cells, our
findings indicate that this improvement of PC3 cell viability
probably is not related to AR expression.
15
50
60
30
0
0
0
Propidium iodide
(a)
4,0
20
10
3,0
2,0
0
0
Propidium iodide
(e)
DMSO (1 day)
20
200
150
100
0
200 400 600 800 1,0 k
Propidium iodide
(i)
20
10
0
200 400 600 800 1,0 k
Propidium iodide
0
100
200 400 600 800 1,0 k
Propidium iodide
(h)
DMSO (1 day) + HG (2 days)
150
Fragmented DNA: 15.4%
G0/G1: 34.7%
S: 29.4%
G2/M: 15.9%
30
(g)
G0/G1: 49.5 %
S: 20.1%
G2/M: 21.1%
10 mM (1 day) + HG (2 days)
Fragmented DNA: 11%
G0/G1: 55.9%
S: 22.3%
G2/M: 9.95%
250
200
G0/G1: 63.7%
S: 12.8%
G2/M: 17.1%
150
100
50
50
0
0
PC3
40
0
200 400 600 800 1,0 k
Propidium iodide
10 mM (1 day)
PC3
Cell count
PC3
60
10
(f)
G0/G1: 70.3 %
S: 12.2%
G2/M: 11%
(d)
5,0
0
200 400 600 800 1,0 k
15
200 400 600 800 1,0 k
Propidium iodide
G0/G1: 37.8%
S: 23.3%
G2/M: 18%
20
1,00
0
0
(c)
22Rv1
30
0
200 400 600 800 1,0 k
Propidium iodide
G0/G1: 39.6%
S: 20.8%
G2/M: 22.1%
5,0
22Rv1
Cell count
22Rv1
40
0
(b)
G0/G1: 41.1%
S: 28.4%
G2/M: 13.4%
40
20
0
200 400 600 800 1,0 k
Propidium iodide
5 mM (1 day)
DMSO (1 day)
50
60
G0/G1 : 24.3%
S: 13.2%
G2/M: 53.1%
30
0
200 400 600 800 1,0 k
90
PNTA1
90
120
60
22Rv1
100
120
G0/G1: 32.2%
S: 14.6%
G2/M: 42.2%
PC3
150
G0/G1: 34.6%
S: 21%
G2/M: 37.7%
PNTA1
Cell count
PNTA1
200
PNTA1
G0/G1: 37.2%
S: 25.7%
G2/M: 27.7%
10 mM (1 day)
DMSO (1 day)
50
0
0
200 400 600 800 1,0 k
Propidium iodide
(j)
0
0 200 400 600 800 1,0 k
Propidium iodide
(k)
0
0 200 400 600 800 1,0 k
Propidium iodide
(l)
Figure 7: Determination of cell cycle analysis by DNA content using flow cytometry. 22Rv1 ((a), (b), (c), and (d)), PNTA1 ((e), (f), (g), and
(h)), and PC3 ((i), (j), (k), and (l)) cells maintained under normal conditions ((a), (e), and (i)), treated with MLT (5 mM or 10 mM) for 1 day
((b), (f), and (j)), incubated with HG medium for 2 days ((c), (g), and (k)), and preincubated with HG medium for 1 day and then exposed
to MLT (5 mM or 10 mM) for an additional 1 day ((d), (h), and (l)). DNA fragmentation: white peaks; G0/G1: blue peaks; S: green areas; and
G2/M: red peaks.
Conflict of Interests
References
The authors declare that no conflict of interests is present in
this paper.
[1] R. J. Reiter and D.-X. Tan, “Melatonin: a multitasking molecule,”
Progress in Brain Research, vol. 181, pp. 127–151, 2010.
Acknowledgments
[2] R. J. Reiter, D.-X. Tan, L. C. Manchester, S. D. Paredes, J. C.
Mayo, and R. M. Sainz, “Melatonin and reproduction revisited,”
Biology of Reproduction, vol. 81, no. 3, pp. 445–456, 2009.
The authors are grateful for the technical assistance of
Mr. Luiz Roberto Faleiros Jr. and Mr. Guilherme Henrique Tamarindo. The work was supported by National
Research Council (CNPq), Fellowship for Rejane M. Góes
(no. 306258/2011-0) and São Paulo State Research Foundation
(FAPESP), Fellowships nos. 2011/19467-0 and 2014/07266-9
for Marina G. Gobbo.
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